Amyloid-like Fibril Formation in an All β-Barrel Protein Involves the Formation of Partially Structured Intermediate(s)

Abstract

In the present study, we demonstrate the thermal induced amyloid formation in a ␤-barrel protein, such as the acidic fibroblast growth factor from Notopthalmus viridescens (nFGF-1). Fibril formation in nFGF-1 is observed to occur maximally at 65°C. Electron microscope analysis of the thermal induced fibrils of nFGF-1 shows that they are filamentous with an average diameter of about 20 nm. X-ray diffraction analysis reveals that the thermal induced fibrils of nFGF-1 have a typical "cross-␤" structure with the ␤-strands perpendicular to the fibril axis. By using a variety of biophysical techniques including multidimensional NMR, we demonstrate that fibril formation involves the formation of a partially structured intermediate(s) in the thermal unfolding pathway of the protein (nFGF-1). Results of the anilino-8-napthalene sulfonate binding experiments indicate that fibril formation occurs due to the coalescence of the protein (in the intermediate state(s))through the solvent-exposed non-polar surface(s). In this study, we also demonstrate that organic osmolytes, such as proline, can efficiently prevent the thermal induced amyloid formation in nFGF-1. Proline is found to stabilize the native conformation of the protein. The addition, proline is observed to increase the cooperativity of the unfolding (native 7 denatured) reaction and consequently decrease the population of the "sticky" thermal equilibrium intermediate(s) responsible for the fibril formation.

Myoglobin forms amyloid fibrils by association of unfolded polypeptide segments

“…It is of note that even unfolded polypeptide chains, such as urea-denatured acidic AMB, can encompass significant amounts of nonrandom conformation (26) and, from the far-UV CD spectrum of heat-unfolded AMB, it is evident that it contains regions that are more structured than the ''classical'' random coil structure of poly(L-lysine) (27). The present observations differ, therefore, from previous analyses of fibril forming proteins that can form well defined partially folded states with ␤-sheet structure (11,12,14,21,28). On the basis of the present data, we propose that the core of AMB amyloid fibrils does not need to be preformed in terms of hydrogen-bonded elements within a monomeric precursor and that partial folding does not represent a general step in amyloid formation, although it may occur in specific cases, such as some ␤-sheet proteins.…”

“…As a further test for the possible presence of partially folded states, we examined AMB by using the dye ANS. This dye has long been utilized to probe for exposure of hydrophobic regions present in partially folded states (20), and a recent study on the formation of amyloid fibrils from acidic fibroblast growth factor demonstrates that this spectroscopic probe can be used under high-temperature conditions (21). As expected, HMB does not bind ANS at 65°C or room temperature, consistent with the absence of stable of stable hydrophobic clusters (data not shown).…”

Systematic Analysis of Aggregates from 38 Kinds of Non Disease-Related Proteins: Identifying the Intrinsic Propensity of Polypeptides to Form Amyloid Fibrils

“…14,15) However, polypeptides tend to form fibrils under various conditions, such as the use of a denaturant, 8,16) high temperature, 17) and extreme pH. 3,4) This sensitivity is one of the intrinsic difficulties in studies of amyloid fibrils.…”

Amyloid-like Fibril Formation in an All β-Barrel Protein

“…7, inset). The fluorescence of the lone tryptophan residue located at position 121 is significantly quenched in the native state of the protein (43)(44)(45)(46). This quenching effect is attributed to the presence of imidazole and pyrrole groups in the vicinity of the indole ring of the tryptophan (Trp-121) residue (31).…”